Endoribonuclease YbeY Is Essential for RNA Processing and Virulence in Pseudomonas aeruginosa

Endoribonuclease YbeY Is Essential for RNA Processing and Virulence in Pseudomonas

aeruginosa

Xia, Yushan; Weng, Yuding; Xu, Congjuan; Wang, Dan; Pan, Xiaolei; Tian, Zhenyang; Xia,

Bin; Li, Haozhou; Chen, Ronghao; Liu, Chang

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Mbio

DOI:

10.1128/mBio.00659-20

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Publication date:

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Xia, Y., Weng, Y., Xu, C., Wang, D., Pan, X., Tian, Z., Xia, B., Li, H., Chen, R., Liu, C., Jin, Y., Bai, F.,

Cheng, Z., Kuipers, O. P., & Wu, W. (2020). Endoribonuclease YbeY Is Essential for RNA Processing and

Virulence in Pseudomonas aeruginosa. Mbio, 11(3), 1-21. [e00659-20].

https://doi.org/10.1128/mBio.00659-20

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Endoribonuclease YbeY Is Essential for RNA Processing and

Virulence in Pseudomonas aeruginosa

Yushan Xia,

_{Yuding Weng,}

_{Congjuan Xu,}

_{Dan Wang,}

_{Xiaolei Pan,}

_{Zhenyang Tian,}

_{Bin Xia,}

_{Haozhou Li,}

Ronghao Chen,

_{Chang Liu,}

_{Yongxin Jin,}

_{Fang Bai,}

_{Zhihui Cheng,}

_{Oscar P. Kuipers,}

_{Weihui Wu}

a_{State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of}

Microbiology, College of Life Sciences, Nankai University, Tianjin, China

b_{Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands}

Yushan Xia and Yuding Weng contributed equally to this work. Author order was determined by the effort in the manuscript submission process.

ABSTRACT

Posttranscriptional regulation plays an essential role in the quick

adap-tation of pathogenic bacteria to host environments, and RNases play key roles in

this process by modifying small RNAs and mRNAs. We find that the Pseudomonas

aeruginosa endonuclease YbeY is required for rRNA processing and the bacterial

vir-ulence in a murine acute pneumonia model. Transcriptomic analyses reveal that

knocking out the ybeY gene results in downregulation of oxidative stress response

genes, including the catalase genes katA and katB. Consistently, the ybeY mutant is

more susceptible to H

O

and neutrophil-mediated killing. Overexpression of katA

restores the bacterial tolerance to H

O

and neutrophil killing as well as virulence.

We further find that the downregulation of the oxidative stress response genes is

due to defective expression of the stationary-phase sigma factor RpoS. We

demon-strate an autoregulatory mechanism of RpoS and find that ybeY mutation increases

the level of a small RNA, ReaL, which directly represses the translation of rpoS

through the 5= UTR of its mRNA and subsequently reduces the expression of the

ox-idative stress response genes. In vitro assays demonstrate direct degradation of ReaL

by YbeY. Deletion of reaL or overexpression of rpoS in the ybeY mutant restores the

bacterial tolerance to oxidative stress and the virulence. We also demonstrate that

YbeZ binds to YbeY and is involved in the 16S rRNA processing and regulation of

reaL and rpoS as well as the bacterial virulence. Overall, our results reveal pleiotropic

roles of YbeY and the YbeY-mediated regulation of rpoS through ReaL.

IMPORTANCE

The increasing bacterial antibiotic resistance imposes a severe threat

to human health. For the development of effective treatment and prevention

strate-gies, it is critical to understand the mechanisms employed by bacteria to grow in

the human body. Posttranscriptional regulation plays an important role in bacterial

adaptation to environmental changes. RNases and small RNAs are key players in this

regulation. In this study, we demonstrate critical roles of the RNase YbeY in the

viru-lence of the pathogenic bacterium Pseudomonas aeruginosa. We further identify the

small RNA ReaL as the direct target of YbeY and elucidate the YbeY-regulated

path-way on the expression of bacterial virulence factors. Our results shed light on the

complex regulatory network of P. aeruginosa and indicate that inference with the

YbeY-mediated regulatory pathway might be a valid strategy for the development of

a novel treatment strategy.

KEYWORDS

endoribonuclease, Pseudomonas aeruginosa, ReaL, RpoS, YbeY

S

uccessful colonization of the host by a pathogenic bacterium depends on efficient

orchestration of global gene expression to quickly adapt to the host in vivo

environment and evade the immune clearance (1). In response to environmental

Citation Xia Y, Weng Y, Xu C, Wang D, Pan X,

Tian Z, Xia B, Li H, Chen R, Liu C, Jin Y, Bai F, Cheng Z, Kuipers OP, Wu W. 2020. Endoribonuclease YbeY is essential for RNA processing and virulence in Pseudomonas aeruginosa. mBio 11:e00659-20.https://doi.org/ 10.1128/mBio.00659-20.

Invited Editor Laurence Rahme, Mass.

General Hospital/Harvard Medical School

Editor Gerald B. Pier, Harvard Medical School Copyright © 2020 Xia et al. This is an

open-access article distributed under the terms of theCreative Commons Attribution 4.0 International license.

Address correspondence to Weihui Wu, wuweihui@nankai.edu.cn.

Received 19 March 2020 Accepted 1 June 2020 Published

RESEARCH ARTICLE

Molecular Biology and Physiology

crossm

®

changes, bacteria control gene expression through transcriptional, posttranscriptional,

and posttranslational mechanisms (2). Compared to transcriptional regulation that

involves RNA synthesis, the posttranscriptional processing provides a way of regulation

that saves time and energy as the mRNA translation and stability are regulated by

RNases and small RNAs (sRNAs) (3–5). By binding to target mRNAs, sRNAs affect

ribosome accessibility or RNase-mediated cleavage. Meanwhile, the processing and

stabilities of sRNAs are under the control of RNases (6–8). Therefore, identification of the

target sRNAs and mRNAs of RNases is essential for the elucidation of the

RNase-mediated regulatory pathways.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes

acute and chronic infections in human (9). Upon infection, phagocytes play an essential

role in the host defense against pathogenic bacteria. One of the major bacterial killing

mechanisms of phagocytes is the generation and release of reactive oxygen species

(ROS) (10, 11). P. aeruginosa harbors a variety of antioxidant enzymes, including

catalases KatA and KatB and alkyl hydroperoxide reductase AphB, AhpC, and AhpF (12,

13). KatA is a constitutively expressed catalase that plays a major role in the bacterial

defense against oxidative stresses and virulence (14–16). Both KatA and KatB are

regulated at the transcriptional level by a variety of factors, including OxyR and the

stationary-phase sigma factor RpoS (17, 18). Mutation of rpoS reduces the expression of

these catalases, leading to increased susceptibility to oxidative stresses and attenuation

of virulence in animal models (17, 19, 20).

RNases have been shown to play important roles in the regulation of virulence

determinants in P. aeruginosa. Previously, we found that the polynucleotide

phosphor-ylase (PNPase) controls the expression of the type III (T3SS) and type VI secretion

systems and pyocin synthesis genes (21, 22). We also found that the PNPase degrades

sRNA P27, which directly controls the translation of the quorum sensing signal synthase

RhlI (23). In P. aeruginosa, PNPase interacts with RNase E and RNA helicase DeaD to form

an RNA degradosome that plays an important role in RNA processing (24). Both RNase

E and DeaD are required for the expression of the T3SS genes (25–27).

YbeY is a highly conserved bacterial RNase that is involved in the maturation of 16S

rRNA, ribosome quality control, regulation of sRNA, and stress responses (28–33). In

pathogenic bacteria, such as enterohemorrhagic Escherichia coli (EHEC), Vibrio cholerae,

and Yersinia enterocolitica, YbeY has been shown to play important roles in bacterial

virulence (29, 34, 35). However, the mechanisms by which YbeY affects bacterial

virulence and stress response remain unclear. Among bacterial species, including P.

aeruginosa, E. coli, and Staphylococcus aureus, the ybeY gene is colocalized with a ybeZ

gene in the same operon (36–38). In E. coli, YbeY has been found to interact with YbeZ

(37), indicating a functional connection between the two proteins. YbeZ contains an

ATP binding and a nucleoside triphosphate hydrolase domain; however, its exact

function remains unknown.

In this study, we demonstrate that the P. aeruginosa endoribonuclease YbeY is

involved in the 16S rRNA maturation, ribosome assembly, and pathogenesis. We further

identify the sRNA ReaL as the target of YebY and elucidate a YbeY-mediated regulatory

pathway that controls the expression of rpoS and oxidative stress response genes. In

addition, we elucidate a posttranscriptional regulatory mechanism of RpoS as well as a

functional connection between YbeZ and YbeY.

RESULTS

YbeY of P. aeruginosa is essential for the 16S rRNA maturation and ribosome

assembly. In P. aeruginosa, gene PA3982 (PA14_12310 in the PA14 genome) encodes

an YbeY homolog. To examine the function of YbeY in P. aeruginosa, we constructed a

ybeY mutant in wild-type PA14. Deletion of the ybeY gene reduced the bacterial growth

rate (Fig. 1A). We then examined its role in the maturation of the 16S rRNA. The ΔybeY

mutant showed an increased proportion of the 16S rRNA precursor, which was restored

to the wild-type level by complementation with a ybeY gene (Fig. 1B). To explore how

YbeY influences the maturation of 16S rRNA, we designed three pairs of real-time PCR

(RT-PCR) primers targeting the middle, 5= starting site, and 3= termination site of the 16S

rRNA, representing the total, 5= immature, and 3= immature 16S rRNA, respectively. In

both the logarithmic and stationary growth phases, the proportion of immature 16S

rRNA in the ΔybeY mutant was higher than that in wild-type PA14, with the 5= immature

Bacterial growth rates. Same numbers of cells of the indicated strains were inoculated in LB. The OD600was

monitored every hour for 12 h. (B) Bacterial cells were cultured in LB to an OD600of 1, followed by RNA isolation.

The 23S and 16S rRNA and the 16S rRNA precursor (17S) were separated by electrophoresis. (C) Top: Schematic diagram of the 16S rRNA precursor. Black arrows indicate the processing sites of RNase III. The red bars represent the regions amplified by real-time PCR. 16S-S, 16S-M, and 16S-T represent the 5= starting site, middle, and 3= termination site of the 16S rRNA, respectively. The bacteria were grown in LB to the OD600of 1 or 3. The bacterial

total RNA was isolated, and the 5= and 3= immature ratios of the 16S rRNA were determined by real-time PCR. ***,

P⬍ 0.001 by Student’s t test. (D) Bacteria were grown to an OD600of 1. The ribosome particles were subjected to

sucrose gradient separation and quantified by UV absorbance at 260 nm.

ratio much higher than the 3= immature ratio (Fig. 1C). We then examined the role of

YbeY in ribosome assembly. Deletion of the ybeY gene reduced the proportion of

assembled 70S ribosome while increasing the proportion of unassembled 30S and 50S

ribosome components (Fig. 1D).

In previous studies, the ybeY mutant was not identified from Tn mutant libraries,

indicating that ybeY might be an essential gene in P. aeruginosa (29, 39). To examine

whether ybeY is essential for P. aeruginosa, we deleted the gene in another two

wild-type strains, PAO1 and PAK. Similar to the PA14 ΔybeY mutant, the ΔybeY mutants

of PAO1 and PAK displayed reduced growth rate and increased proportion of 16S rRNA

precursor (see Fig. S1 in the supplemental material). These results suggest that YbeY

plays important roles in the rRNA processing and growth of P. aeruginosa.

To confirm that the P. aeruginosa ybeY (PA3982) gene is a true ortholog of the ybeY

gene in E. coli, we complemented the PA14 ΔybeY mutant with an E. coli ybeY gene,

which restored the bacterial growth rate and maturation of the 16S rRNA (Fig. S2A, B,

and C). Consistent with previous studies, the E. coli ybeY mutant was more susceptible

to heat shock and oxidative stresses (such as H

O

) (29). Complementation of the E. coli

ybeY mutant with the ybeY gene from PA14 restored the bacterial resistance to heat

shock and H

O

(Fig. S2D and E).

Previous studies in E. coli and Bacillus subtilis revealed that the amino acid residues

R56 and H112 are critical for the function of YbeY (28, 40, 41). To determine the

importance of these conserved amino acid residues in the P. aeruginosa YbeY, we

replaced the R56 or H112 with alanine (A). Neither of the alleles restored the maturation

of the 16S RNA and the growth rate of the ΔybeY mutant (Fig. 2B and C), indicating the

essentialities of these residues in the function of the P. aeruginosa YbeY.

YbeZ binds to YbeY and contributes to the processing of the 16S rRNA. In the

P. aeruginosa genome, ybeY (PA3982) is predicted to be in the same operon with ybeZ

(PA3981) and ybeX (PA3983). RT-PCR results confirmed the cotranscription of these

three genes (Fig. S3). A previous study in E. coli revealed the interaction between YbeY

and YbeZ (37), suggesting that YbeZ might function as a partner of YbeY. To test

whether YbeY and YbeZ are functionally connected in P. aeruginosa, we performed a

pulldown assay. Our results revealed active interaction between YbeY and YbeZ

(Fig. 3A). Next, we examined the biological function of YbeZ. Mutation of ybeZ also

reduced the bacterial growth rate (Fig. S4A) and maturation of the 16S rRNA, but to a

lesser extent compared to the ybeY mutation (Fig. 3B to D). For example, at the OD

of 1.0, the 16S rRNA 5= and 3= immature ratios of the ΔybeZ mutant were approximately

13- and 60-fold higher than those in the wild-type PA14, respectively, whereas the

corresponding differences between the ΔybeY mutant and the wild-type PA14 were

approximately 43- and 100-fold (Fig. 3C and D). However, mutation of ybeZ did not

affect the assembly of the ribosome (Fig. S4B). These results indicate that YbeY and

YbeZ might form a complex that processes the 16S rRNA precursor, with YbeY playing

a major role. Accordingly, we focused our following studies on the functions of YbeY.

YbeY is required for the virulence of P. aeruginosa in a murine acute

pneumo-nia model. To examine the role of YbeY in the virulence of P. aeruginosa, we utilized

a murine acute pneumonia model. Mutation of ybeY reduced the bacterial load by

approximately 600-fold, which was restored by the complementation with a wild-type

ybeY gene (Fig. 4A).

To understand the mechanism of YbeY-mediated regulation on the bacterial

viru-lence, we performed transcriptome analyses. Expression of 313 genes was altered by

the mutation of ybeY (Fig. 4B; Table S2). Of note, genes involved in the oxidative stress

response were downregulated in the ΔybeY mutant, including the catalase genes katA

and katB, the alkyl hydroperoxide reductase gene aphC, and the superoxide dismutase

gene sodB (Table 1). The catalases play critical roles during P. aeruginosa infection. KatA

is a constitutive and housekeeping catalase produced by P. aeruginosa and plays a

critical role in the bacterial tolerance to oxidative stresses and virulence (42, 43),

whereas the expression of katB is induced by oxidative stresses (15). Mutation of yebY

reduced the mRNA levels of katA and katB in the presence and absence of H

O

, which

were restored by complementation with the yebY gene (Fig. 5A and B). Consistent with

the gene expression pattern, the total catalase activity of the ΔybeY mutant was lower

than that of the wild-type PA14, which was restored by overexpression of katA (Fig. 5C).

aeruginosa and E. coli YbeY. Identical amino acids are indicated by dark blue; the similar amino acids are indicated

by lighter blue. The conserved R56 and H112 are indicated by red boxes. (B) Bacterial growth rates in LB. Same numbers of cells of the indicated strains were inoculated in LB. The OD600was monitored every hour for 12 h. (C)

The bacteria were cultured in LB to an OD600of 1, followed by RNA isolation. The 23S and 16S rRNA and the 16S

rRNA precursor (17S) were separated by electrophoresis.

In the acute pneumonia model, the neutrophil plays an important role in the host

clearance of the invading P. aeruginosa (12). Generation of ROS is one of the major

bacterium-killing mechanisms by the neutrophil (11, 12). The downregulation of the

oxidative response genes might render the ΔybeY mutant more susceptible to the

neutrophil-mediated killing. Indeed, after incubation with neutrophils differentiated

from HL60 cells (dHL60), the survival rate of the ΔybeY mutant was significantly lower

than that of the wild-type strain (Fig. 5D). Supplementation of the ROS scavenger

molecule N-acetylcysteine (NAC) or overexpression of katA restored the bacterial

survival rate (Fig. 5D). In addition, the ΔybeY mutant was more susceptible to H

O

treatment and overexpression of the katA gene restored the bacterial survival rate

(Fig. 5E). Furthermore, overexpression of katA in the ΔybeY mutant restored the

bacterial load in the acute pneumonia model (Fig. 5F) without affecting the bacterial

growth rate in LB (Fig. S5). These results indicate that the defective bacterial response

to ROS contributes to the attenuated virulence of the ΔybeY mutant.

FIG 3 YbeZ binds to YbeY and contributes to the processing of the 16S rRNA. (A) Examination of the interaction between YbeY and YbeZ

by a pulldown assay. Cell lysates containing the YbeY-His were incubated with the resin bound with GST or YbeZ-GST for 2 h. The beads were washed three times with the cell lysis buffer. The bound proteins were eluted with GSH and subjected to Western blotting. (B) Bacterial cells were cultured in LB to an OD600of 1, followed by RNA isolation. The 23S and 16S rRNA and the 16S rRNA precursor (17S)

were separated by electrophoresis. (C and D) The 5= immature (C) and 3= immature (D) ratio of 16S rRNA was determined by real-time PCR.**, P⬍ 0.01; ***, P ⬍ 0.001 by Student’s t test.

YbeY controls the oxidative stress response genes through RpoS. In P.

aerugi-nosa, the transcriptional regulator OxyR controls the expression of the catalase and the

alkyl hydroperoxide reductase genes in response to oxidative stresses (44). In addition,

the ATP-dependent helicase RecG facilitates the binding of OxyR to the promoters of

target genes (45, 46). In the ΔybeY mutant, the expression levels of oxyR, recG, and ahpB

(alkyl hydroperoxide reductase) were similar to those in the wild-type PA14 in the

presence and absence of H

O

(Fig. S6). The aphC mRNA level in the ΔybeY mutant was

lower in the absence of H

O

but increased to a similar level as that in PA14 in the

presence of H

O

. These results indicate that YbeY might not control the expression of

katA/B through OxyR and RecG.

Besides OxyR and RecG, the alternative sigma factor RpoS had been demonstrated

to affect the expression of katA/B and the bacterial tolerance to H

O

(18–20). Our

RNA-seq result revealed downregulation of the rpoS gene in the ΔybeY mutant

(Ta-ble 1), which was confirmed by real-time PCR (Fig. 6A). Overexpression of rpoS in the

ΔybeY mutant restored the expression of katA (Fig. 6B) and the total catalase activity in

the presence and absence of H

O

(Fig. 6C) as well as the bacterial tolerance to H

O

(Fig. 6D). In addition, mutation of rpoS did not affect the expression of ybeY at either the

logarithmic or stationary growth phase (Fig. S7). In combination, these results

demon-strate that YbeY controls the expression of katA through RpoS.

YbeY controls the expression of rpoS at the posttranscriptional level. We then

explored the mechanism of YbeY-mediated regulation of rpoS. The transcription of rpoS

was examined using a transcriptional fusion between the rpoS promoter and a

infected intranasally with the indicated strains. At 12 h postinfection, lungs from the infected mice were isolated. The bacterial loads were determined by serial dilution and plating.***, P⬍ 0.001 by Student’s

t test. (B) RNA-seq results. Values reported as log(RPKM) of the genes with more than 2-fold difference

in expression between the ΔybeY mutant and the wild-type PA14.

TABLE 1 mRNA levels of oxidative response genes in the ΔybeY mutant compared to

those in wild-type PA14

Gene name Product

Fold change

⌬ybeY/PA14 P value

katA Catalase 0.03 3.31E⫺07

katB Catalase 0.11 9.11E⫺12

ahpC Alkyl hydroperoxide reductase 0.11 4.88E⫺07

sodB Superoxide dismutase 0.28 7.76E⫺04

rpoS RNA polymerase sigma factor RpoS 0.34 9.11E⫺12

FIG 5 Mutation of ybeY reduces the bacterial response to oxidative stress and virulence. Wild-type PA14, ΔybeY mutant,

and the complemented strain were grown in LB to an OD600of 1 and then incubated with or without 2 mM H2O2for

30 min. (A and B) The relative mRNA levels of katA (A) and katB (B) were determined by real-time PCR. Results represent means⫾ SD. (C) The indicated strains were grown in LB to an OD600of 1 and then incubated with or without 2 mM H2O2

for 30 min. The bacteria were collected by centrifugation, and the cells were broken by sonication. The total intracellular catalase activity was measured using a catalase assay kit. (D) The indicated strains were incubated with dHL60 cells at an MOI of 5 in HBSS or HBSS with 80 mM N-acetylcysteine for 3 h. The bacterial survival rates were determined by plating. (E) The indicated strains were grown in LB to an OD600of 1. The cells were washed three times with PBS and then incubated

with 10, 20, or 50 mM H2O2for 30 min. The bacteria were collected by centrifugation and resuspended with fresh LB, and

the survival rates were determined by plating. (F) Mice were infected intranasally with the indicated strains. At 12 h postinfection, lungs from the infected mice were isolated. The bacterial loads were determined by serial dilution and plating.*, P⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 by Student’s t test.

moterless lacZ reporter gene (P

-lacZ).

␤-Galactosidase assay revealed a reduced rpoS

promoter activity in the ΔybeY mutant (Fig. 7A). A previous ChIP-seq analysis indicated

a possible binding of RpoS to its own promoter, suggesting an autoregulation (47). To

verify whether RpoS controls its own expression, we utilized an rpoS::Tn mutant from

the PA14 transposon insertion mutant library (48). Mutation in rpoS reduced the LacZ

expression from P

-lacZ, which was restored by overexpression of an rpoS gene

driven by an exogenous tac promoter (Fig. 7A). An EMSA demonstrated that RpoS

indeed bound to its own promoter but not the coding sequence (Fig. 7B). Collectively,

these results suggest an autoregulation of rpoS.

To elucidate the mechanism of YebY-mediated regulation of rpoS, we overexpressed

the rpoS gene by a constitutively active tac promoter in the ΔybeY mutant, which

restored the expression of the P

-lacZ (Fig. 7A). These results raise the possibility that

the lower rpoS promoter activity in the ΔybeY mutant might be due to a deficiency in

the translation of the rpoS mRNA. To examine the translation of rpoS, we constructed

a series of C-terminal Flag-tagged rpoS fusions (rpoS-Flag) driven by an exogenous

arabinose-inducible promoter (P

) with various lengths of the rpoS 5= UTR, resulting

FIG 6 YbeY controls the bacterial response to oxidative stresses through rpoS. (A) PA14 and the ΔybeY mutant

were cultured in LB to an OD600of 1 or 3. The relative mRNA levels of rpoS were determined by real-time PCR.

Results represent means⫾ SD. (B and C) The indicated strains were grown in LB to an OD600of 1 and then

incubated with or without 2 mM H2O2for 30 min. (B) The relative mRNA levels of katA were determined by real-time

PCR. Results represent means⫾ SD. (C) The bacteria were collected by centrifugation, and the cells were broken by sonication. The total intracellular catalase activity was measured using a catalase assay kit. (D) The indicated strains were grown in LB to an OD600of 1, and the cells were washed three times with PBS and then incubated with

10, 20, or 50 mM H2O2for 30 min. The bacteria were collected by centrifugation and resuspended with fresh LB,

and the survival rates were determined by plating. ns, not significant;*, P⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 by Student’s t test.

in rpoS(F1)-FLAG, rpoS(F2)-FLAG, and rpoS(F3)-FLAG (Fig. 7C). The translation of rpoS

was reduced in the ΔybeY mutant even when the 5= UTR was truncated to 25

nucleotides upstream of the start codon. However, when the 25-nucleotide sequence

was replaced by an exogenous ribosome binding sequence from vector plasmid

pET28a, the RpoS-FLAG protein levels were similar between wild-type PA14 and the

ΔybeY mutant (Fig. 7C and D). In combination, these results demonstrate that YbeY

affects the translation of rpoS.

YbeY controls the rpoS translation through sRNA ReaL. In P. aeruginosa, two

sRNAs, namely, RgsA and ReaL, repress the rpoS translation by targeting to the

⫺25 to

FIG 7 YbeY affects the translation of rpoS through a 25-nucleotide sequence in the 5= UTR. (A) The indicated strains containing PrpoS-lacZ

transcriptional fusion were cultured in LB to an OD600of 1 or 3. The bacteria were collected and subjected to the␤-galactosidase activity

assay. ns, not significant;*, P⬍ 0.05; ***, P ⬍ 0.001 by Student’s t test. (B) The 6⫻His-tagged RpoS protein was expressed in E. coli and purified through Ni-NTA affinity chromatography. Pro-rpoS and In-rpoS represent the fragments of the rpoS promoter and coding regions, respectively. The location of the fragments was indicated by arrows. One hundred nanograms purified DNA fragment was incubated with the indicated amounts of RpoS protein for 30 min, followed by electrophoresis in a nondenatured polyacrylamide gel. The arrow indicates the DNA-protein complex. (C) Diagrams of C-terminal Flag-tagged rpoS fusions (rpoS-Flag) driven by an exogenous arabinose-inducible promoter (PBAD) with various lengths of the rpoS 5= UTR. RBS, ribosome binding sequence from the vector pET28a. (D) PA14 and its ΔybeY

mutant strain containing the individual RpoS translation fusions were cultured in LB to an OD600of 1 and then incubated with 0.2%

⫹27 (relative to the start codon) and the Shine-Dalgarno (SD) regions (⫺13 to ⫺6) of

the rpoS mRNA, respectively (3, 49, 50). In the ΔybeY mutant, rgsA was downregulated

whereas reaL was upregulated (Fig. 8A), indicating a possible involvement of ReaL in

the repression of the rpoS translation. Indeed, deletion of reaL in the ΔybeY mutant

restored the translation of the P

-driven rpoS-FLAG while the mRNA levels of the

rpoS-FLAG were similar among the strains (Fig. 8B).

To further verify that the repression of rpoS is due to the upregulation of reaL, we

mutated the predicted binding site of ReaL (3, 49, 50) in the rpoS(F1)-FLAG,

rpoS(F2)-FLAG, and rpoS(F3)-FLAG (Fig. 8C), which resulted in similar expression levels of the

RpoS-FLAG in wild-type PA14 and the ΔybeY mutant (Fig. 8C). Meanwhile, deletion of

reaL in the ΔybeY mutant background restored the mRNA levels of katA and rpoS

(Fig. 8D and E). Overexpression of reaL in the double-knockout mutant reduced the

rpoS mRNA level, whereas overexpression of ybeY resulted in a higher rpoS mRNA level

than that in the wild-type PA14 (Fig. 8E). These results indicate that the high level of

ReaL in the ΔybeY mutant contributes to the downregulation of rpoS and that YbeY

might positively regulate rpoS through other mechanisms.

YbeY directly degrades ReaL. To explore the mechanism of the upregulation of

ReaL, we constructed a transcriptional fusion of the reaL promoter with a promoterless

lacZ gene (P

-lacZ). The LacZ levels were similar in wild-type PA14 and the ΔybeY

mutant (Fig. 9A), indicating a similar transcriptional level of the sRNA. We then

examined the stability of ReaL. Following blockage of the RNA synthesis by rifampin,

the ReaL level dropped quickly in wild-type PA14, whereas a slower reduction was

observed in the ΔybeY mutant (Fig. 9B). Meanwhile, the mRNA level of RpsL (a

ribosomal subunit) dropped at a similar rate in the two strains (Fig. 9C). These results

suggest that the higher level of ReaL in the ΔybeY mutant is likely due to its increased

stability.

To address whether ReaL is a direct target of YbeY, we tested whether ReaL can

interact with YbeY by EMSA in the presence of an RNase inhibitor. As shown in Fig. 9D,

YbeY retarded the migration of ReaL, indicating a direct interaction. Next, we

investi-gated whether YbeY can degrade ReaL directly. As shown in Fig. 9E and F, the presence

of Mn

_{significantly increased the degradation of the ReaL RNA by YbeY, which is}

consistent with previous reports that YbeY is a metal-dependent endoribonuclease (1,

6, 10). Furthermore, mutation of the conserved residues R56 and H112 or both of them

in YbeY reduced the degradation efficacy (Fig. 9E and F).

Downregulation of rpoS by ReaL plays a major role in the attenuated virulence

of the

⌬ybeY mutant. Next, we examined whether the upregulation of ReaL

contrib-utes to the defective oxidative response and attenuated virulence of the ΔybeY mutant.

Deletion of reaL in the ΔybeY mutant restored the total catalase activity (Fig. 10A) as

well as the bacterial tolerance to H

O

(Fig. 10B), whereas deletion of reaL in the

wild-type PA14 did not affect these phenotypes. In addition, overexpression of rpoS or

deletion of reaL in the ΔybeY mutant restored the bacterial survival rate after incubation

with dHL60 cells (Fig. 10C). We further examined the bacterial virulence in the acute

pneumonia model. Overexpression of rpoS or deletion of reaL in the ΔybeY mutant

restored the bacterial loads in the lungs (Fig. 10D), whereas deletion of reaL in the

wild-type PA14 did not affect the bacterial loads in the lungs (date not shown). Deletion

of reaL in the ΔybeY mutant did not affect the bacterial growth rate, while

overexpres-sion of RpoS slightly increased the growth rate (Fig. S5). In combination, these results

indicate that the ReaL-mediated downregulation of RpoS and subsequent defective

oxidative stress response contribute to the attenuated virulence of the ΔybeY mutant.

Mutation of ybeZ results in similar phenotypes as those of the

⌬ybeY mutant.

As we found that YbeZ binds to YbeY and is involved in the maturation of the 16S rRNA,

it is likely that YbeZ plays similar roles as YbeY. Indeed, mutation of ybeZ resulted in

upregulation of reaL and downregulation of rpoS and katA (Fig. 11A). Consistently, the

ΔybeZ mutant was more susceptible to H

O

and displayed attenuated virulence in the

acute pneumonia model (Fig. 11B and C). These results suggest that YbeY and YbeZ are

FIG 8 YbeY controls the translation of RpoS through ReaL. (A) Wild-type PA14, the ΔybeY mutant, and the complemented strain were grown in

LB to an OD600of 1. The relative levels of the sRNAs reaL and rgsA were determined by real-time PCR. Results represent means⫾ SD. ***, P ⬍

0.001 by Student’s t test. (B) PA14, the ΔybeY mutant, and the ΔybeY ΔreaL mutant containing the RpoS(F1)-FLAG translational fusion were cultured in LB to an OD600of 1 and then incubated with 0.2%L-arabinose for 60 min. The bacteria were collected for Western blot analysis. The

relative mRNA levels of the rpoS-Flag were determined by real-time PCR using the primers matching to the Flag tag RNA. The results represent means⫾ SD. ns, not significant. (C) The potential base-pairing between ReaL and the 5= UTR is indicated by asterisks. The mutated nucleotides are underlined. PA14 and the ΔybeY mutant containing the indicated rpoS-Flag fusions were cultured in LB to an OD600of 1 and then incubated

with 0.2%L-arabinose for 60 min. The bacteria were collected for Western blot analysis. (D and E) The indicated strains were grown in LB to an OD600of 1. The relative mRNA levels of katA (D) and rpoS (E) were determined by real-time PCR. The results represent means⫾ SD. **, P ⬍ 0.01

likely to function together in the regulation of reaL, rpoS, and oxidative stress response

genes.

DISCUSSION

YbeY is a highly conserved bacterial RNase that is involved in the 16S rRNA

maturation, ribosome assembly, virulence, and stress responses (28–33). In previous

studies, the ybeY mutant was not identified from P. aeruginosa transposon mutant

libraries. Thus, ybeY was predicted to be an essential gene (29, 39). In this study, we

were able to delete ybeY in the backgrounds of PA14, PAO1, and PAK. However, it took

at least 2 days for the ΔybeY mutant to form visible colonies on the LB plate. We suspect

that the growth defect might explain why the ybeY mutant was not identified from the

Tn mutant population. Mutation of ybeY resulted in defective processing of the 16S

rRNA, defective response to oxidative stresses, and attenuated virulence in the murine

acute pneumonia model. In addition, we found that the ybeY mutant was more

susceptible to heat shock (45°C, data not shown).

FIG 9 YbeY directly degrades ReaL. (A) PA14 and the ΔybeY mutant containing the PreaL-lacZ transcriptional fusion were cultured in LB to an OD600of 1 or 3.

The bacteria were collected and subjected to the␤-galactosidase activity assay. ns, not significant. (B and C) Degradation of ReaL (B) and the rpsL mRNA (C) in wild-type PA14 and the ΔybeY mutant. Bacterial cells were treated with rifampin to stop the transcription. At indicated time points, the bacteria were collected and mixed with equal numbers of gfp-expressing E. coli cells. Total RNA was isolated, and the relative RNA levels were determined by real-time PCR. The gfp mRNA in each sample was used as the internal control for normalization. (D) The 6⫻His-tagged YbeY protein was expressed in E. coli and purified through Ni-NTA affinity chromatography. One hundred nanograms purified RNA transcript was incubated with the indicated amounts of the YbeY protein with an RNase inhibitor for 30 min on ice, followed by electrophoresis in a nondenatured polyacrylamide gel. The arrow indicates the RNA-protein complex. (E and F) RNA degradation by wild-type (E) and the mutated (F) YbeY. Purified GFP, wild-type YbeY, and those with indicated mutations were incubated with 100 ng purified ReaL at 37°C for 30 min with indicated concentrations of MnCl2. Then the samples were analyzed by electrophoresis in a nondenatured polyacrylamide gel. The

RNA bands were visualized by staining with Gel-red (Biotium).

Previous studies in E. coli identified YbeY as a metal-dependent hydrolase, and the

three-dimensional crystal structure of the YbeY revealed a conserved metal ion binding

pocket (40). Deletion of ybeY in E. coli reduces protein translation efficiency by affecting

the 30S ribosome subunit. Davies et al. reported that YbeY is involved in the maturation

of 16S rRNA (30). Jacob et al. demonstrated that YbeY is a single-stranded RNA (ssRNA)

specific endoribonuclease and plays key roles in the ribosome quality control and 16S

FIG 10 YbeY regulates the bacterial response to oxidative stresses and virulence through ReaL and RpoS. (A) The indicated strains were grown

in LB to an OD600of 1 and then incubated with or without 2 mM H2O2for 30 min. The bacteria were collected by centrifugation, and the cells

were broken by sonication. The total intracellular catalase activity was measured using a catalase assay kit. (B) The indicated strains were grown in LB to an OD600of 1, and the cells were washed three times with PBS and then incubated with 10, 20, or 50 mM H2O2for 30 min. The bacteria

were collected by centrifugation and resuspended with fresh LB, and the survival rates were determined by plating. (C) The indicated strains were incubated with dHL60 cells at an MOI of 5 in HBSS for 3 h. The bacterial survival rates were determined by plating. (D) Mice were infected intranasally with the indicated strains. At 12 h postinfection, lungs from the infected mice were isolated. The bacterial loads were determined by serial dilution and plating. ns, not significant;**, P⬍ 0.01; ***, P ⬍ 0.001 by Student’s t test.

FIG 11 Roles of YbeZ in the bacterial response to oxidative stress and virulence. (A) Wild-type PA14, the

ΔybeZ mutant, and the complemented strain were grown in LB to an OD600of 1. The relative RNA levels

of katA, rpoS, and reaL were determined by real-time PCR. Results represent means ⫾ SD. (B) The indicated strains were grown in LB to an OD600of 1. Then the cells were washed three times with PBS

and incubated with 10, 20, or 50 mM H2O2for 30 min. The bacteria were collected by centrifugation and

resuspended with fresh LB, and the survival rates were determined by plating. (C) Mice were infected

(Continued on next page)

rRNA maturation together with RNase R in E. coli (28). A structural model of the E. coli

YbeY revealed a positively charged cavity similar to the middle domain of Argonaute

(AGO) proteins involved in RNA silencing in eukaryotes (51). Recent studies in E. coli,

Sinorhizobium meliloti, and Vibrio cholerae demonstrated that the defect in YbeY results

in aberrant expression of small RNAs (sRNAs) and the corresponding target mRNAs (29,

51, 52). The YbeY purified from S. meliloti displays a metal-dependent endoribonuclease

activity that cleaves both ssRNA and double-stranded RNA (dsRNA) substrates (33).

A previous bacterial two-hybrid analysis revealed that YbeY interacts with the

ribosomal protein S11, Era, Der, SpoT, and YbeZ in E. coli (37). The interaction between

YbeY and S11 is required for the maturation of the 16S rRNA (37). Overexpression of the

GTPase gene era in a ΔybeY mutant improves the 16S rRNA maturation and 70S

ribosome assembly (53). SpoT plays an important role in the bacterial stringent

re-sponse by controlling the homeostasis of the alarmone molecule (p)ppGpp (54). Of

note, (p)ppGpp controls the bacterial growth rate by suppressing the rRNA production

(55). Further studies are warranted to explore whether YbeY affects the (p)ppGpp level

and thus the bacterial stringent response. The GTPase Der (double Era-like GTPase)

contains two GTP-binding domains. Studies in E. coli demonstrated its essentiality in the

biogenesis of the 50S ribosomal subunit (56). In bacteria, the colocalization of ybeZ and

ybeY in an operon is highly conserved (36–38). Here, we demonstrated the interaction

between YbeY and YbeZ in P. aeruginosa and found that mutation of ybeZ resulted in

similar phenotypes as the ybeY mutant. Further studies are needed to understand the

exact function of YbeZ.

Here, we found that YbeY affects the expression of RpoS. A comparison of the

transcription profiles between the ybeY mutant and an rpoS mutant revealed that 49

genes displayed similar expression patterns in the two mutants compared to the

wild-type strain (see Table S2 in the supplemental material) (57). For example, previous

studies revealed that the P. aeruginosa lectin PA-IL coding gene lecA and the

two-component response regulator gene pprB are under the direct regulation of RpoS (58,

59). Mutation of rpoS resulted in downregulation of lecA, pprB, and genes regulated by

the PprB, including the tad locus (PA4297-PA4305) and the type IVb pilin gene flp (59).

Mutation of ybeY also reduced the expression of those genes (Table S2). However, 7

genes were oppositely expressed in the ybeY mutant and the rpoS mutant (Table S2),

and many other genes displayed different expression patterns in the two mutants.

These results indicate that YbeY controls global gene expression through multiple

pathways.

In P. aeruginosa, RpoS controls the expression of the catalase genes katA/B (18);

however, no RpoS binding sequence has been identified in the promoter regions of the

two genes, indicating an indirect regulation (47). Overexpression of rpoS in the ΔybeY

mutant restored the expression levels of katA (Fig. 6) and sodB (data not shown).

However, overexpression of rpoS only restores the expression level of katB in the ΔybeY

mutant in the absence of H

O

, but not in the presence of H

O

(data not shown). The

RpoS-mediated regulatory pathways on katA, katB, and sodB warrant further studies.

Upon invading host, the bacteria encounter phagocytes that kill the bacteria mainly

through ROS, phagocytosis, antimicrobial peptides, and hydrolases (12, 60, 61). Bacteria

survive the phagocyte-generated ROS by producing superoxide dismutase and

cata-lases (13, 62). Our in vitro infection assay revealed that the ybeY mutant was more

susceptible to neutrophils. Neutralization of ROS by NAC restored the bacterial survival

rate, indicating that the hypersusceptibility to neutrophil is mainly due to defective

detoxification of the ROS. Therefore, the downregulation of katA might partially

con-tribute to the attenuated virulence of the ybeY mutant. Meanwhile, RpoS controls

multiple stress response genes and affects the quorum sensing systems (57). The

FIG 11 Legend (Continued)

intranasally with the indicated strains. At 12 h postinfection, lungs from the infected mice were isolated. The bacterial loads were determined by serial dilution and plating.*, P_{⬍ 0.05; ***, P ⬍ 0.001 by Student’s}

defective processing of rRNA might also affect the bacterial adaptation to the host

environment and expression of virulence factors. Comparing the gene expression

profile between the ybeY mutant and the wild-type strain during infection might shed

light on the roles of YbeY in the bacterial virulence.

Overall, our study reveals an important physiological role of YbeY in the RNA processing

in P. aeruginosa. Further studies are required to understand how the expression and activity

of YbeY are regulated, particularly under environmental stresses.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains, plasmids, and primers used in this study are

listed in Table S1 in the supplemental material. Bacteria were cultured in the L-broth (63) medium at 37°C with agitation at 200 rpm. Antibiotics were used at the following concentrations: for E. coli, gentamicin 10␮g ml⫺1, tetracycline 10␮g ml⫺1, kanamycin 50␮g ml⫺1, ampicillin 100␮g ml⫺1; for P. aeruginosa, tetracycline 50␮g ml⫺1_{, gentamicin 50␮g ml}⫺1_{, rifampin 100␮g ml}⫺1_{, carbenicillin 150␮g ml}⫺1_.

Chromosomal gene mutations were generated as described previously (64). To construct a ybeY deletion mutant in P. aeruginosa, a 945-bp fragment and a 1,306-bp fragment that are upstream and downstream of the ybeY coding region, respectively, were amplified by PCR using PA14 chromosome as the template and primers listed in Table S1. The fragments were cloned into the plasmid pEX18TC. The resulting plasmid was transferred into E. coli S17 and then transferred into P. aeruginosa by conjunction. Single crossover mutants were selected on 50␮g ml⫺1_{tetracycline and 50␮g ml}⫺1_{kanamycin (to kill the E. coli}

donor strain), and double crossover mutants were selected by growth on LB plates containing 7.5% sucrose. The ybeY deletion mutant was screened by PCR with the primers YbeY-L and YbeY-R (Table S1). In the ybeY deletion mutant, a 421-bp fragment was deleted from the 483-bp coding region of the ybeY gene.

Transcriptome sequencing and analysis. Bacteria were cultured in LB at 37°C to the stationary

phase (OD600⫽ 3). Total RNA was extracted by the RNA prep Pure Cell/Bacteria kit (Tiangen Biotec,

Beijing, China). Sequencing and analysis services were performed by the Suzhou Genewiz as previously described (65).

Real-time PCR. Bacterial cells were cultured under indicated conditions. Total bacterial RNA was

extracted by an RNA prep Pure Cell/Bacteria kit (Tiangen Biotec, Beijing, China). cDNAs were synthesized using random primers and reverse transcriptase (TaKaRa, Dalian, China). Real-time PCR was performed with the SYBR II Green supermix (TaKaRa, Dalian, China). The ribosomal gene rpsL or PA1805 was used as the internal control (66).

rRNA analysis. Bacteria were cultured in LB at 37°C until late logarithmic phase (OD600⫽ 1). Total

RNA was isolated with the RNA prep Pure Cell/Bacteria kit (Tiangen Biotec, Beijing, China). One microgram of the total RNA was mixed with an RNA loading buffer (TaKaRa, Dalian, China) and incubated at 65°C for 10 min, followed by incubation on ice for 10 min. Then the 16S and 23S rRNAs were separated by electrophoresis on a gel made of 0.9% Synergel (BioWorld, USA) and 0.7% agarose in TAE as described previously (67).

Ribosome separation by sucrose gradients. Separation of P. aeruginosa ribosomal particles was

performed as previously described (41) with minor modifications. Bacteria were grown in LB at 37°C to an OD600of 1. One liter of the bacterial cells was collected and resuspended in 40 ml precooled Buffer

A (10 mM Tris–HCl, 10 mM MgCl2, 100 mM NH4Cl, 6 mM ␤-mercaptoethanol, pH 7.5) with 10 ␮g/ml

DNase I. The bacterial cells were lysed by a French press (600 MPa), and the cell debris was removed by centrifugation at 12,000⫻ g for 30 min at 4°C. One milliliter of the supernatant was loaded onto a 10% to 40% sucrose gradient in buffer A, followed by centrifugation at 36,000 rpm for 3 h at 4°C in an SW41 rotor (Beckman). The stratified sucrose gradient solutions were collected, and the RNA contents were quantified by UV absorbance at 260 nm.

Pulldown assay. The ybeY and ybeZ coding regions were amplified by PCR using PA14 chromosome

DNA as the template, and the primers are listed in Table S1. A 6⫻His tag coding sequence was included in the downstream primer of the ybeY gene (Table S1), thus resulting in a C-terminal 6⫻His-tagged ybeY (ybeY-His). The amplified ybeY gene was cloned into the plasmid pMMB67EH. The ybeZ gene was cloned into the plasmid pET41a, resulting in a translational fusion with the gst gene at the C terminus. E. coli strain BL21 carrying the gst gene or ybeZ-gst fusion gene was cultured at 37°C to an OD600of 0.4 to 0.6.

Expression of the YbeZ-GST was induced by 1 mM IPTG for 4 h, and then the bacteria were collected and resuspended in a lysis buffer (1 M Na2HPO4, 1 M NaH2PO4, 0.3 M NaCl, pH 8.0), followed by sonication.

The cell debris was removed by centrifugation at 12,000_{⫻ g for 20 min at 4°C. The cell lysate containing} GST or YbeZ-GST protein was incubated with the GST tag purification resin (Beyotime Biotechnology, Shanghai, China) for 2 h at 4°C and then washed three times with the lysis buffer. PA14 carrying the

ybeY-His fusion gene was grown in LB to an OD600of 0.6, followed by induction by 1 mM IPTG for 4 h.

The bacteria were resuspended in the lysis buffer and lysed by sonication. The cell lysate was then incubated with the GST or YbeZ-GST bound resin at 4°C for 2 h. The resin was washed three times with the lysis buffer. The bound proteins were then eluted with 60 mM reduced glutathione (GSH) in an elution buffer provided by the purification kit (Beyotime Biotechnology, Shanghai, China) and subjected to Western blot analysis.

␤-Galactosidase assay. Bacterial cells were grown in LB at 37°C until the OD600reached 1. An

0.5-ml portion of the bacterial culture was collected and resuspended in 1.5 ml Z buffer (50 mM

␤-mercaptoethanol, 60 mM Na2HPO4, 60 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0). The

␤-galactosidase activity was determined as previously described (68).

H2O2susceptibility assay. Bacteria were cultured to an OD600of 1 and then washed three times with

PBS and resuspended in PBS. The bacteria were treated with 10, 20, or 50 mM H2O2at 37°C for 30 min.

The live bacterial numbers were determined by serial dilution and plating. The survival rate was calculated by comparing the live bacterial number after the H2O2 treatment with that before the

treatment.

Catalase activity assay. Bacteria were cultured to an OD600of 1 and then incubated with or without

2 mM H2O2for 30 min. The bacteria were collected by centrifugation, and the cells were broken by

sonication. The total intracellular catalase activity was measured using a catalase assay kit (Beyotime, Shanghai, China). The total bacterial protein concentrations were quantified by a BCA analysis (Beyotime, Shanghai, China) for calibration.

HL60 differentiation and killing assay. HL60 cells were cultured in RPMI 1640 medium (HyClone,

USA), with streptomycin (100 mg/ml), penicillin G (100 U/ml), and 10% (vol/vol) thermally inactivated fetal bovine serum (Gibco, Australia) at 37°C with 5% CO2. The HL60 cells were diluted to 4.5⫻ 105

cells/ml and cultured in the differentiation medium (RPMI 1640 medium, 20% [vol/vol] heat-inactivated fetal bovine serum, 1.3% dimethyl sulfoxide [Sigma, USA]) for 6 to 7 days (20). The differentiated HL60 cells (designated dHL60) were diluted to 1⫻ 107_{cells/ml in warm HBSS, and 100}␮l cell suspension was

added to each well of a 96-well plate. Then the cells were infected with bacteria at an MOI of 10 and incubated at 37°C for 3 h. The live bacterial numbers were determined by serial dilution and plating. The survival rate was calculated by comparing the numbers of live bacterial cells after incubation with or without the dHL60 cells.

RNA stability analysis. P. aeruginosa strains were cultured in LB at 37°C to an OD600of 1. The

bacterial cultures were then treated with 100␮g/ml rifampin to stop the transcription. At the indicated time points, the bacterial cells were collected and mixed with the same number of E. coli cells expressing a gfp gene. Total RNA was purified, and the levels of reaL and rpsL were determined by real-time PCR. The gfp mRNA levels were used as the internal control for normalization.

In vitro transcription and RNA gel mobility shift assay. The in vitro transcription of sRNA was

performed as previously described (22). The sRNA ReaL was synthesized using the Riboprobe System-T7 (Promega) from PCR product amplified from PA14 chromosomal DNA with the primers listed in Table S1 according to the manufacturer’s instructions. The RNA was purified by isopropanol precipitation and refolded by heating at 90°C for 10 min and then cooling down naturally at room temperature for 30 min. One hundred nanograms of the purified RNA was mixed with indicated amounts of purified YbeY or GFP in the binding buffer (10 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 50 mM KCl, 10% glycerol, 1 U recombinant

RNase inhibitor [TaKaRa]) and incubated on ice for 30 min. Fifteen microliters of each sample was loaded onto a nondenaturing 8% polyacrylamide gel. The electrophoresis was performed at 100 V for 150 min in 0.5⫻ TBE buffer on ice. The RNA bands were visualized after staining with Gel-red (Biotium) for 10 min.

In vitro RNA degradation assay. The sRNA ReaL was synthesized using a Riboprobe System-T7

(Promega) from the PCR product amplified from PA14 chromosomal DNA with the primers listed in Table S1. The RNA was purified by isopropanol precipitation and refolded by heating at 90°C for 10 min and then cooling down naturally at room temperature for 30 min. One hundred nanograms of the purified RNA was mixed with indicated amounts of the purified YbeY or GFP protein in the activity buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 10% glycerol, and the indicated concentrations of MnCl2) and

incubated at 37°C for 30 min. Fifteen microliters of each sample was loaded onto a nondenaturing 8% polyacrylamide gel. Electrophoresis was performed at 100 V for 150 min in 0.5⫻ TBE buffer on ice. The RNA was visualized after staining with Gel-red (Biotium) for 10 min.

Murine acute pneumonia model. The animal infection experiments described in this study were

performed following the National and Nankai University guidelines on the use of animals in research. The protocol with the permit number NK-04-2012 was approved by the animal care and use committee of the College of Life Sciences, Nankai University. The infection was performed as previously described (69). Briefly, bacteria were cultured overnight and then diluted at 1:100 in fresh LB and grown at 37°C with agitation until OD600reached 1. The bacterial cells were harvested by centrifugation, washed once with

PBS, and resuspended in PBS at the concentration of 2⫻ 108_{CFU/ml. Each 6- to 8-week-old female}

BALB/c mouse (Vital River, Beijing, China) was anesthetized by an intraperitoneal injection of 100␮l 7.5% chloral hydrate, followed by an intranasal inoculation of 20␮l of the bacterial suspension, resulting in 4⫻ 106_{CFU per mouse. The mice were sacrificed 12 h postinfection, and the lungs were dissected and}

subjected to homogenization. The number of bacteria in each lung was determined by plating.

Data availability. The transcriptome (RNA sequencing) data that support the findings of this study

have been deposited in the NCBI Sequence Read Archive (SRA) with the accession codePRJNA574019.

SUPPLEMENTAL MATERIAL

Supplemental material is available online only.

FIG S1, PDF file, 0.2 MB.

FIG S2, PDF file, 0.2 MB.

FIG S3, PDF file, 0.3 MB.

FIG S4, PDF file, 0.1 MB.

FIG S5, PDF file, 0.1 MB.

FIG S6, PDF file, 0.1 MB.

FIG S7, PDF file, 0.1 MB.

TABLE S1, DOCX file, 0.04 MB.

TABLE S2, DOCX file, 0.03 MB.

ACKNOWLEDGMENTS

We thank Silke Bonsing-Vedelaar of University of Groningen Faculty of Science and

Engineering for the ybeY knockout E. coli strain.

This work was supported by National Key Research and Development Project of

China (2017YFE0125600), National Science Foundation of China (31670130, 31970680,

31870130, and 81670766), the Tianjin Municipal Science and Technology Commission

(19JCYBJC24700), and the program of China Scholarships Council (no. 201906200035).

The funders had no role in study design, data collection and interpretation, or the

decision to submit the work for publication.

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